Download GENETIC TECHNOLOGY

GENETIC TECHNOLOGY
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Recombinant DNA technology
Use
of laboratory techniques to isolate and
manipulate fragments of DNA
Recombinant DNA contains DNA from 2 or
more sources
Once inside a host cell, recombinant
molecules are replicated to produce
identical copies or clones
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Gene cloning (Molecular Cloning)
Molecular cloning refers to the procedure
of isolating a defined DNA sequence and
obtaining multiple copies of it in vivo.
Cloning is frequently employed to amplify
DNA fragments containing genes.
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Step 1 in gene cloning
Vector DNA as a
carrier for the DNA
segment to be cloned
When a vector is
introduced into a living
cell, it can replicate
making many copies
Common vectors are
plasmid or viral
Also need the gene of
interest from
chromosomal DNA
digest
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Step 2
Insert chromosomal DNA into vector
Cut DNA using restriction enzymes or restriction
endonucleases
Made
naturally by bacteria as protection against
bacteriophages
Cuts at specific known restriction sites
Most restriction sites palindromic
May produce sticky ends
DNA ligase must be used to permanently link DNA
Result may be
Recircularized
vector with no gene of interest inserted
Recombinant vector with gene of interest inserted
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Vector carries a selectable marker
Presence
of antibiotics selects for cells
expressing ampR gene – contained in plasmid
ampR gene codes for b-lactamase that
degrades ampicillin, which normally kills
bacteria
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Step 3
Goal for recombinant vector to be taken up
by bacteria
Transformation – plasmid vector
Transfection – viral vector
After treatment, only cells with the plasmid
will grow on plates treated with ampicillin
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To eliminate recircularized vectors from further
examination, lacZ gene part of vector
Insertion of chromosomal DNA disrupts lacZ
gene
lacZ codes for b-galactosidase which cleaves
colorless X-Gal into a blue dye
Recircularized plasmids will form blue colonies
Recombinant vectors will form white colonies
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DNA library
Treatment of chromosomal DNA with
restriction enzymes yields thousands of
different fragments
DNA library- collection of many
recombinant vectors each with a fragment
of chromosomal DNA
2 types of common DNA libraries
Genomic
– inserts derived from chromosomal
DNA
cDNA – use reverse transcriptase to make
DNA from mRNA of interest (complementary
DNA) - lacks introns so simpler to use
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Use colony hybridization to identify
colonies with gene of interest
Master
plate with bacterial cells containing
recombinant vector with different pieces of
chromosomal DNA
Use single-stranded DNA probe
Dark spots correspond to colonies with gene
of interest
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Blotting Methods Can Identify Gene Families
Southern blotting can detect the presence of a
particular gene within a mixture of many
chromosomal fragments separated on a gel
Another common use of Southern blotting is to
identify gene families
Two or more genes are derived from the same ancestral
gene
The members of a gene family are homologous,
having similar but not identical DNA sequences
Distinguish homologous members of a gene family within
a single species or identify homologous genes among
different species
Electrophoresis
Separates macromolecules based on charge
and size
DNA is negative – moves toward positive electrode
Smaller fragments move faster/farther than large
fragments
Bands can be stained or probed
Southern blotting of a gel
Locations where probe binds appear dark on xray film
High stringency – detects close match between
probe and chromosomal DNA
Low stringency – detects homologous genes
Polymerase chain reaction (PCR)
Copy DNA without vectors and host cells
Goal to make many copies of DNA in a defined
region
Uses high concentration of two primers that are
complementary to sequences at the ends of the
DNA region to be amplified, deoxynucleoside
triphosphates (dNTPs), and a heat-stable form
of DNA polymerase called Taq polymerase
Sample of DNA taken through repeated cycles
of denaturation, annealing and synthesis
Thermocycler
automates this process
After 30 cycles of amplification, a DNA sample
will increase 230-fold
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Genomics
Refers
to the molecular analysis of the entire
genome of a species
2 types
Structural – direct DNA analysis – mapping
Functional – studying expression – which genes
turned on or off in particular cells
Proteomics
Used
to identify and study groups of proteins
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BACs and YACs
Most plasmid and viral vectors accept a
few to tens of thousands of nucleotides
Bacterial artificial chromosomes (BAC)
can contain up to 500,000 bp
Yeast artificial chromosomes (YAC) can
take up to 2 million nucleotides
Used in first phases of mapping –
determining relative locations of inserted
chromosomal pieces on intact
chromosome
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Contig
Series
of clones
that contain
overlapping pieces
of chromosomal
DNA
Overlapping
regions allow
researchers to
identify their order
along the
chromosome
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DNA sequencing
Determines base sequence of DNA
Dideoxy chain-termination method or dideoxy
sequencing
Dideoxynucleoside
triphosphates (ddNTPs) are
missing the 3’ –OH group and will terminate the chain
4 tubes with many copies of single stranded DNA of
interest
Each tube has a different radiolabelled dNTP
DNA
polymerase will make complementary strand
until ddNTP inserted and chain terminates
After electrophoresis, DNA sequence can be read by
reading which base is at the end of the DNA strand
Procedure has been automated using
fluorescent dyes
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Microarray or gene chip
Used in functional genomics
Monitors expression of thousands of genes
simultaneously
Small slide dotted with many different short
sequences of single-stranded DNA from known
genes
RNA isolated from sample cells used to make
fluorescently labeled cDNA
cDNA that is complementary to the DNA in the
microarray will hybridize
Measure fluorescence intensity
Identifies genes that have been transcribed in a
particular cell under a given set of circumstances
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Proteomics
Entire collection of a species’ proteins
Due to gene regulation, any given cell will
produce only a subset of its proteome
What proteins are made depends on what
type of cell it is, stage of development, and
environmental conditions
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2D gel electrophoresis
Can separate thousands of different
proteins in a cell extract
Sample of proteins loaded onto tube
shaped gel
Isoelectric focusing – separates proteins
according to their net charge at a given pH
Tube gel then placed on slab gel that
separates proteins by molecular mass
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Mass spectroscopy
Proteins can be identified by their unique amino
acid sequence
Tandem mass spectrometry uses 2
spectrometers
First
measures mass of a given peptide
Second measures mass after the peptide has been
digested into smaller fragments
Researcher could determine the possible codon
sequences that could encode such a peptide
More
than one sequence is possible due to
degeneracy of genetic code
Using computer software, entire genomic sequence
scanned
Can locate match between codon sequence and
specific gene
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Biotechnology
Technologies that involve the use of living
organisms to benefit humans
Use began about 1200 years ago with
livestock domestication
More recently associated with molecular
genetics
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Insulin
In 1982, US FDA approved sale of human
insulin made by recombinant bacteria
Prior to 1982, insulin isolated from cattle
Some
people developed allergies and had to use
cadaver insulin
Insulin composed of 2 polypeptides – A and B
A
and B coding sequence inserted into E.coli
Fusion proteins extracted and b-galactosidase
removed
Purified A and B chain mixed to form functional
protein
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Bioremediation
Use of microorganisms to decrease
pollutants in the environment
Enzymes produced by microorganism can
alter or transform toxic pollutant structure
– biotransformation
May degrade toxic form into less complex,
nontoxic metabolites – biodegradation
1980, first patented recombinant
microorganism
Not
a commercial success
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Transgenics
An organism that carries genes introduced using
molecular techniques such as gene cloning
Genetically
modified organisms (GMOs)
Gene replacement – cloned gene recombines
with normal gene on a chromosome
Only
1 of 2 copies replaced creating hetrozygote
Heterozygotes can be crossed to yield homozygotes
Gene knockout – if cloned gene is a mutation
that inactivates function, homozygote will not
have gene function
Application in studying human disease
Used
as models to study disease
Used to test effect of therapies
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Molecular pharming
Production of medically important proteins
in livestock mammary glands
Certain proteins more likely to function
when expressed in mammals
Post-translational
modification
Degraded or improperly folded in bacteria
High yield in cows
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Molecular pharming
Strategy to clone gene next to the
promoter of a gene specifically expressed
in mammary cells
Vector injected into oocyte, fertilized and
implanted
Milk containing hormone purified for
protein
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Transgenic plants
Somewhat easier because plant cells are totipotent
Cloned genes can be introduced into somatic tissue and
entire plant regenerated with hormonal treatments
Agrobacterium tumefaciens naturally infects plant cells
and causes tumors
Ti plasmid modified to introduce cloned genes
Contains Ti plasmid that integrates into host chromosome
Codes for plant growth hormones that form crown gall tumor
KanR used as a selectable marker for kanamycin resistance
Contain convenient restriction sites
Transformed cells plated on media with kanamycin (kills
nontransformed cells) and carbenicillin (kills
Agrobacterium)
Transgenic plants are approved for human consumption
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Cloning mammals
Identical twins are genetic clones from a
single fertilized egg that split early in
development
Plants can be cloned from somatic cells
1996, Dolly is the first cloned lamb
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Mammary cells from adult
sheep removed
Fused diploid mammary
cell with enucleated
sheep oocyte
Zygote implanted
Dolly and donor were
almost genetically
identical
Same set of genes
Minor differences due
to differences in
mitochondrial DNA
and maternal effect
genes
Achieved in several
mammal species
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DNA fingerprinting/ DNA profiling
Identifies particular individual using properties of
their DNA
Chromosomal DNA produces series of bands on
a gel
Unique pattern of bands used
Automated using PCR to amplify short tandem
repeat sequences (STRs)
Uses
Identify
different species
Forensics – 1986 first use in US court system
Paternity testing
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Blaese and Colleagues Performed the First
Gene Therapy to Treat ADA Deficiency
Gene therapy introduces cloned genes into living
cells to cure disease
More than 4,000 diseases involve a single gene
abnormality
Adenosine deaminase deficiency prevents proper
metabolism of nucleosides
Deoxyadenosine accumulates and results in
destruction an B and T cells
Leads to severe combined immunodeficiency
syndrome (SCID)
Fatal
at early age (1 or 2 years old)
3 treatment approaches
Bone
marrow transplant
Purified ADA enzyme
Gene therapy
Gene therapy
September
14, 1990
Remove lymphocytes from girl
Treat with retroviral vector containing ADA
gene
Return cells to her bloodstream
Results suggest that this first gene therapy
trial may offer benefit but patients also
treated with ADA